Baccharis é um dos gêneros mais expressivos em Asteraceae, com 360 espécies registradas nas Américas, especialmente na América do Sul. No Brasil são 115 espécies endêmicas, cuja popularidade é difundida na medicina popular em decorrência de espécies medicinais conhecidas como carquejas, e empregadas centenariamente por seus efeitos hepatoprotetor, em desordens renais, no tratamento da diabetes, entre outros. Essas propriedades são decorrentes da elevada diversidade química do gênero, com destaque aos terpenos e compostos fenólicos. O objetivo do trabalho foi caracterizar as substâncias fenólicas presentes nas partes aéreas de Baccharis oblongifolia (Ruiz & Pav.) Pers. e determinar as atividades antirradicalar e citotóxica dos componentes, assim como contribuir na elucidação da constituição química em plantas dioicas, característica marcante do gênero estudado. Após obtenção dos extratos e fases de partição de B. oblongifolia, quinze substâncias foram identificadas através dos espectros de RMN 1H, RMN 13C e técnicas bidimensionais, ou por comparação com padrões comerciais e/ou previamente isolados pelo grupo de pesquisa. Entre os componentes foram identificados dez derivados do ácido clorogênico, os ácidos 5-cafeoilquínico, 3,4-dicafeoilquínico, 3,5-dicafeoilquínico, 4,5-dicafeoilquínico e 3,4,5-tricafeoilquínico, e seus respectivos ésteres metílicos, o ácido cafeico, e quatro flavonoides, rutina (quercetina-3-O-rutinosídeo), nicotiflorina (caempferol-3-O-rutinosídeo), quercetina-3-O-β-[2\'\'-O-(E)-cafeoil]-rutinosídeo e caempferol-3-O-β-[2\'\'-O-(E)-cafeoil]-rutinosídeo. Os dois últimos flavonoides são substâncias inéditas em literatura. As atividades antirradicalar e citotóxica foram avaliadas para todos os extratos, fases de partição e substâncias isoladas obtendo-se resultados promissores para os ensaios antirradicalares. Não houve variação qualitativa entre os metabólitos produzidos nos indivíduos dioicos de B. oblongifolia, / Baccharis is one of the most expressive genera in Asteraceae, with 360 species found in the Americas, especially in South America. In Brazil there are 115 endemic species, whose popularity is widespread in folk medicine due to medicinal species known as \"carquejas\". For centuries these plants have been used for their hepatoprotective effects, in renal disorders, in the treatment of diabetes, among others. These properties are due to the high chemical diversity found in the genus, with emphasis on terpenoids and phenolic compounds. The objectives of this work were: 1. to characterize the phenolic compounds produced by aerial parts of Baccharis oblongifolia (Ruiz & Pav.) Pers.; 2. to determine the antiradical and cytotoxic activities of the components; 3. to contribute to elucidation of the chemical constitution in dioecious plants, a remarkable characteristic of the genus. After obtaining extracts and partitioning phases from B. oblongifolia, fifteen compounds were identified through 1H NMR, 13C NMR and two-dimensional spectra, or by comparison with commercial standards and/or previously isolated compounds by our research group. Among the components, were identified ten chlorogenic acid derivatives, 5-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and 3,4,5-tricaffeoylquinic acid, and their respective methyl esters, caffeic acid, and four flavonoids, rutin (quercetin-3-O-rutinoside), nicotiflorin (kaempferol-3-O-glucoside), quercetin-3-O-β-[2\'\'-(E)-caffeoyl]-rutinoside and kaempferol-3-O-β-[2\'\'-O-(E)-caffeoyl]-rutinoside. The last two flavonoids are new compounds. The antiradical and cytotoxic activities were evaluated for extracts, partition phases and isolated compounds. Promising results were obtained for the antiradical assays. There was no qualitative variation between the metabolites produced in the dioecious individuals of B. oblongifolia

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-09-27T13:47:07Z No. of bitstreams: 1 ANA ZILDA CEOLIN COLPO 2017.pdf: 6048240 bytes, checksum: 62bfa1f3557cbe81c077e11ac86b15a3 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-09-27T13:47:29Z (GMT) No. of bitstreams: 1 ANA ZILDA CEOLIN COLPO 2017.pdf: 6048240 bytes, checksum: 62bfa1f3557cbe81c077e11ac86b15a3 (MD5) / Made available in DSpace on 2017-09-27T13:47:30Z (GMT). No. of bitstreams: 1 ANA ZILDA CEOLIN COLPO 2017.pdf: 6048240 bytes, checksum: 62bfa1f3557cbe81c077e11ac86b15a3 (MD5) Previous issue date: 2017-08-09 / O consumo de bebidas a base de erva-mate é parte dos hábitos e costumes de populações do sul do Brasil, Argentina e Uruguai. Seu consumo é um símbolo cultural vinculado à sociabilidade e demonstrações de cordialidade. Aprimorar o conhecimento sobre a associação de seu consumo com a saúde humana pode levar a formulação de paradoxos de grande relevância econômica e social. Assim, esta pesquisa teve como objetivos: i) avaliar a capacidade dos extratos de erva-mate modularem o equilíbrio redox a nível sistêmico em Drosophila melanogaster e em nível de sistema nervoso central (SNC) em ratos; ii) observar como os extratos podem afetar eventos genotóxicos relacionados a perda da heterozigose em asas de Drosophila melanogaster. Dietas acrescidas de colesterol foram usadas como indutoras de danos no estudo com moscas da fruta e imobilização no estudo com animais. Para avaliar a relação do extrato com mutagênese e antimutagênese empregou-se o Teste de Mutação e Recombinação Somáticas (SMART). Além disso, no delineamento experimental deste trabalho projetou-se o uso de extratos (mates) obtidos mimetizando a forma tradicional de consumo, assim nas abordagens com moscas da fruta empregaram-se extratos obtidos por extrações sequenciais. Com animais, por limitações impostas pelo experimento, empregou-se o extrato bruto obtido de forma convencional. Nesta vertente também se incluiu a avaliação dos efeitos do composto majoritário em extratos de erva-mate, o ácido clorogênico (CGA). Os resultados demostram que extratos de erva-mate, em Drosophila melanogaster expostas à dieta hipercolesterolêmica, são capazes de aumentar significativamente a capacidade de resistir a estresse induzido melhorando a homeostase redox. O que foi constatado pela redução dos níveis de produtos da peroxidação lipídica, recuperação da atividade enzimática da glutationa S-transferase (GST) e aumento do tempo de vida dos insetos. Nas regiões cerebrais córtex (CTX), hipocampo (HIP) e corpo estriado (STR) a indução de estresse por imobilização produz danos a lipídeos e proteínas. Mate e CGA foram capazes de atenuar esses prejuízos, demostrando seu potencial de melhorar a resistência a estresse induzido, efeito possivelmente mediado por sua capacidade antioxidante. Para completar, os resultados obtidos para a atividade genotóxica mostraram que mate não induz danos em asas de Drosophila, quer seja por processos mutacionais ou recombinatórios. Dados relativos à avaliação da antigenotoxicidade indicaram que os extratos reduziram a frequência de manchas mutantes em co-tratamento. No entanto, podem apresentar efeitos genotóxicos quando administrados após danos, isto pode ser resultado de sua ação na modulação de funções detoxificantes no organismo, relacionadas principalmente ao citocromo P450 (CYP). Com base em dados da literatura estas informações levam a suposição que compostos fenólicos podem provocar inibição do CYP reduzindo a metabolização e a eliminação de drogas, o que produz acumulação e aumento da toxicidade. Conclui-se, por meio do uso dos diferentes modelos experimentais e das propostas deste estudo, que a erva-mate possui significativa atividade antioxidante e não produz efeito genotóxico per se, mas sua relação com antigenotoxidade e metabolização de xenobióticos precisa ser confirmada por estudos aprofundados nesta abordagem. / The consumption of yerba-mate based beverages is part of the habits of the population from South Brazil, Argentina and Uruguay. Its consumption is a cultural symbol linked to sociability and demonstrations of cordiality. Improving the knowledge about the association of yerba-mate consumption with human health can lead to the creation of great economic and social relevant paradoxes. Thus, this research had as objectives: i) to evaluate the capacity of yerba-mate extracts on modulating the redox balance at systemic level in Drosophila melanogaster and at the central nervous system (CNS) in rats; ii) to observe how the extracts can affect genotoxic events related to heterozygosity loss on Drosophila melanogaster wings. Diets with high cholesterol added were used as damage inducers on the study with fruit flies and immobilization with animals. The Somatic Mutation and Recombination Test (SMART) was used to evaluate the relation of the extract with mutagenesis and antimutagenesis. In addition, in the experimental design of this work, were used extracts (mates) obtained mimicking the traditional form of consumption. Thus, in the approaches with fruit flies, extracts obtained by sequential extractions were used. Because of the limitations imposed by the experiment, the crude extract obtained by the conventional manner was used in the animals. In this strand, the evaluation of the effects of the major compound in yerba-mate extracts, the chlorogenic acid (CGA), was also included. The results showed that yerba-mate extracts, in Drosophila melanogaster exposed to hypercholesterolemic diet, are able to increase significantly the capacity to resist the induced stress, improving the redox homeostasis. This was evidenced by the reduction of the levels of lipid peroxidation products, glutathione S-transferase (GST) activity recovery and increase of the insects’ lifespan. In the brain regions: cortex (CTX), hippocampus (HIP) and striatum (STR), the induction of immobilization stress produces damage to lipids and proteins. Mate and CGA were able to mitigate these damages, demonstrating their potential to improve induced stress resistance. This effect was possibly mediated by its antioxidant capacity. To complete, the results obtained for the genotoxic activity showed that mate does not induce damage in Drosophila wings, either by mutational or recombinant processes. Data for the evaluation of the antigenotoxicity indicated that the extracts reduced the frequency of mutant stains in co-treatment. However, they can present genotoxic effects when given after damage. This may be due to its action on modulating detoxifying functions in the organism, related mainly to the P450 cytochrome (CYP). Based on the literature data, this information lead to the assumption that phenolic compounds may cause CYP inhibition, reducing metabolization and drug elimination, which produces accumulation and increased toxicity. Were possible to conclude, by using different experimental models and the study proposals, that yerba-mate has significant antioxidant activity and does not produce genotoxic effect per se, but its relation with antigenotoxicity and xenobiotic metabolization needs to be confirmed by deepened studies focused on this approach.

A pele possui um grande número de mecanismos de defesa antioxidante. No entanto, a exposição prolongada à radiação ultravioleta (RUV), é capaz de aumentar a concentração de espécies reativas de oxigênio (EROs) provocando um desequilíbrio antioxidante/oxidante. A geração de EROs pode levar ao fotoenvelhecimento e doenças, como o câncer. A administração tópica de antioxidantes é uma maneira eficiente de enriquecer o sistema protetor cutâneo endógeno e, assim, uma estratégia para reduzir os danos oxidativos causados pela RUV à pele. Entretanto, para fornecer a proteção adequada à pele, estas substâncias devem ultrapassar a barreira imposta pelo estrato córneo. O extrato de Lychnophora salicifolia Mart. possui compostos com atividades antioxidante, anti-inflamatória e analgésica, como o ácido clorogênico e a vicenina-2, sendo, portanto, um possível candidato à agente protetor dos danos induzidos pela radiação na pele. Assim, o objetivo do presente trabalho foi avaliar o potencial fotoquimiopreventivo deste extrato e do ácido clorogênico, bem como de formulações contendo o extrato e a substância isolada encapsulados em lipossomas. Para atingir tal objetivo, foram realizados experimentos in vitro e in vivo de atividade antioxidante e eficácia fotoquimiopreventiva para comprovação do potencial antioxidante e anti-inflamatório, estudos de liberação e estudos de penetração cutânea das formulações utilizando pele humana e pele de orelha de porco. Os resultados obtidos demonstraram que o extrato possui atividade antioxidante in vitro e baixa citotoxicidade. A incorporação do extrato e da substância isolada em lipossomas foi bem sucedida, uma vez que estes demonstraram uma boa estabilidade físico-química e aumentaram a penetração dos compostos na pele de camundongos sem pelos. As formulações contendo lipossomas permitiram que os compostos do extrato e o ácido clorogênico isolado atravessassem o estrato córneo chegando até epiderme e derme na pele humana ex vivo. O acúmulo de partículas nos folículos pilosos parece não exercer influencia neste processo. Os extrato foi capaz de sequestrar EROs intracelulares e evitar a depleção do antioxidante endógeno GSH tanto em cultura de células como nos experimentos in vivo. Também foi observada a inibição in vivo da metaloproteinase de matriz-9 (MMP-9), enzima ativada pela RUV e que tem a capacidade de degradar os componentes da matriz extracelular da pele. Além disso, o extrato apresentou atividade anti-inflamatória superior à do ácido clorogênico, verificada pela diminuição dos níveis de citocinas pró-inflamatórias (IL-1?, IL-6 e TNF-?) e da atividade da enzima mieloperoxidase (MPO) in vivo. Portanto, o extrato parece ser um agente fotoquimiopreventivo promissor para uso tópico e, as formulações desenvolvidas, um sistema viável para liberação destas substâncias na pele. / The skin has several defense mechanisms to avoid oxidant damages. However, a prolonged exposition under ultraviolet radiation (UVR) may increase reactive oxygen species (ROS) concentration causing antioxidant/oxidant imbalance. ROS generation can induce photoaging and skin cancer. Topical administration of antioxidants is an efficient way to fortify the endogenous protection system and to reduce the oxidative damage caused by UVR. Nevertheless, to be effective, these compounds need to pass through the skin barrier imposed by stratum corneum. Lychnophora salicifolia Mart. extract has antioxidant, antiinflammatory and analgesic compounds, such as chlorogenic acid and vicenin-2, and it may be a skin protector agent against UVR damage. Therefore, the aim of this study was to evaluate the photochemopreventive potential of this extract and chlorogenic acid, as well as their formulation containing liposomes. In vitro and in vivo tests were performed to prove antioxidant and antiinflammatory activity. Formulation release and skin penetration studies were also carried out in human and porcine ear skin. The results showed that the extract has antioxidant activity in vitro and low cytotoxicity. The extract and the isolated compound were successfully entrapped in liposomes, demonstrating a good physic and chemical stability and increasing the skin penetration in hairless mice. The formulations containing liposomes allowed that the extract compounds and chlorogenic acid passed through the stratum corneum and reached epidermis and dermis in human skin ex vivo. The hair follicle uptake of the particles do not influenced the penetration process. The extract was able to scavenge intracellular ROS and avoid the depletion of the endogenous GSH both in cell culture and in vivo, even as it inhibited matrix metalloproteinase 9 (MMP-9), enzyme activated by UVR that degrades skin matrix extracellular components. The extract also presented larger antiinflammatory properties than chlorogenic acid, decreasing the cytokines (IL-1?, IL-6 and TNF-?) levels and the mieloperoxidase activity in vivo. Therefore, the results suggest the potential applicability of the extract as a photochemopreventive agent to topical use and the developed formulations seem to be a viable system to release these compounds to the skin.

The phenolic compounds as food components represent the largest group of secondary metabolites in plant foods. The phenolic compounds, e.g. chlorogenic acid (CQA), are susceptible to oxidation by enzymes specially, polyphenol oxidase (PPO) and at alkaline conditions. Both enzymatic and non-enzymatic oxidations occur in the presence of oxygen and produce quinone, which normally further react with other quinone to produce colored compounds (dimers), as well as is capable of undergoing a nucleophilic addition to proteins. The interactions of proteins with the phenolic compounds have received considerable attention in the recent years where, plant phenolic compounds have drawn increasing attention due to their antioxidant properties and their noticeable effects in the prevention of various oxidative stress associated diseases. Green coffee beans are one of the richest sources of chlorogenic acids. Therefore, a green coffee extract would provide an eligible food relevant source for phenolic compounds for modification of proteins. The interaction between 5-CQA and amino acid lysine showed decrease in both free CQA and amino acid groups and only a slight effect on the antioxidative capacity depending on the reaction time was found. Furthermore, this interaction showed a large number of intermediary substances of low intensities. The reaction of lysine with 5-CQA in a model system initially leads to formation of 3-CQA and 4-CQA (both are isomers of 5-CQA), oxidation giving rise to the formation of a dimer which subsequently forms an adduct with lysine to finally result in a benzacridine derivative as reported and confirmed with the aid of HPLC coupled with ESI-MSn. The benzacridine derivative containing a trihydroxy structural element, was found to be yellow, being very reactive with oxygen yielding semiquinone and quinone type of products with characteristic green colors. Finally, the optimal conditions for this interaction as assessed by both the loss of CQA and free amino groups of lysine can be given at pH 7 and 25°C, the interaction increasing with incubation time and depending also on the amount of tyrosinase present. Green coffee bean has a higher diversity and content of phenolics, where besides the CQA isomers and their esters, other conjugates like feruloylquinic acids were also identified, thus documenting differences in phenolic profiles for the two coffee types (Coffea arabica and Coffea robusta). Coffee proteins are modified by interactions with phenolic compounds during the extraction, where those from C. arabica are more susceptible to these interactions compared to C. robusta, and the polyphenol oxidase activity seems to be a crucial factor for the formation of these addition products. Moreover, In-gel digestion combined with MALDI-TOF-MS revealed that the most reactive and susceptible protein fractions to covalent reactions are the α-chains of the 11S storage protein. Thus, based on these results and those supplied by other research groups, a tentative list of possible adduct structures was derived. The diversity of the different CQA derivatives present in green coffee beans complicates the series of reactions occurring, providing a broad palette of reaction products. These interactions influence the properties of protein, where they exposed changes in the solubility and hydrophobicity of proteins compared to faba bean proteins (as control). Modification of milk whey protein products (primarily b-lactoglobulin) with coffee specific phenolics and commercial CQA under enzymatic and alkaline conditions seems to be affecting their chemical, structural and functional properties, where both modifications led to reduced free amino-,thiol groups and tryptophan content. We propose that the disulfide-thiol exchange in the C-terminus of b-lactoglobulin may be initiated by the redox conditions provided in the presence of CQA. The protein structure b-lactoglobulin thereupon becomes more disordered as simulated by molecular dynamic calculation. This unfolding process may additionally be supported by the reaction of the CQA at the proposed sites of modification of -amino groups of lysine (K77, K91, K138, K47) and the thiol group of cysteine (C121). These covalent modifications also decreased the solubility and hydrophobicity of b-lactoglobulin, moreover they provide modified protein samples with a high antioxidative power, thermally more stable as reflected by a higher Td, require less amount of energy to unfold and when emulsified with lutein esters, exhibit their higher stability against UV light. The MALDI-TOF and SDS-PAGE results revealed that proteins treated at alkaline conditions were more strongly modified than those treated under enzymatic conditions. Finally, the results showed a slight change in emulsifying properties of modified proteins. / Für die Verbesserung von Nahrungsmitteleigenschaften können Modifikationen an verschiedenen Inhaltsstoffen vorgenommen werden. Beispielsweise werden bereits Proteine miteinander verknüpft und bilden sogenannte „Crosslinks“ oder vernetzte Biomoleküle. Diese werden für die Herstellung fester, viskoelastischer Produkte, die zum Verdicken als auch zum Stabilisieren von Emulsionen oder Schäumen eingesetzt werden, genutzt. Da die Verbraucher sich Zunehmens mit gesundheitsfördernden Lebensmitteln befassen, ist das Einbringen von gesundheitsfördernden Inhaltsstoffen wie z.B. phenolische Verbindungen, immer mehr in den Fokus der Forschung gerückt. Demnach ist das wissenschaftliche Bestreben phenolische Verbindungen in die Vernetzung von Proteinen mit einzubeziehen und deren positive Wirkungen (antioxidativ) auszunutzen, vorteilhaft. Als Phenole werden Verbindungen bezeichnet, die eine oder mehrere Hydroxygruppen am Benzolring aufweisen. Phenole liegen in der Enolform vor, da diese, bedingt durch den Erhalt des aromatischen Benzolringes, energetisch begünstigt ist. Kaffeesäure ist eine Hydroxyzimtsäure und in Kaffeebohnen zu finden. Der am häufigsten anzutreffende Ester besteht aus Kaffee- und Chinasäure. Der einfachste Vertreter ist die Chlorogensäure (5-Caffeoylchinasäure, 5-CQA), die in vielen Pflanzenteilen enthalten ist. Chlorogensäure und ihre Derivate besitzen ebenfalls antioxidative Eigenschaften. Zusätzlich wirken sie auf Enzyme, die an entzündlichen- oder allergischen Reaktion teilnehmen, inhibierend. Während Verarbeitungs- und Lagerungsprozessen können phenolische Komponenten pflanzlicher Lebensmittel mit den Aminosäuren der Proteine in Lebensmitteln reagieren. Solche Reaktionen können die physikalisch-chemischen Eigenschaften von Proteinen verändern und deren ernährungsphysiologische Wertigkeit vermindern. Proteine weisen verschiedene reaktive Seitengruppen (Sulfhydryl-, Hydroxyl-, Aminogruppen) auf, mit denen sie über kovalente und nicht-kovalente Wechselwirkungen mit Phenolen Verbindungen eingehen können. Zu den nicht-kovalenten Verbindungen gehören u. a. Wasserstoffbrückenbindungen, hydrophobe Wechselwirkungen und Ionenbindungen. Phenole (z.B. Chlorogensäuren) können bei Anwesenheit von Sauerstoff enzymatisch bzw. nichtenzymatisch oxidiert werden. Die Reaktionsprodukte (Chinone) bilden anschließend mit reaktiven Thiol- bzw. Aminogruppen von Proteinen Addukte. Die Erfassung dieser verschiedenen Facetten von Interaktionen stellt somit die primäre Forschungsaufgabe im Rahmen dieser Arbeit. Die primäre Aufgabe der vorliegenden Arbeit besteht demzufolge in der Etablierung der Analysen- und der Charakterisierungsmöglichkeiten solcher Wechselwirkungen (Bindung) pflanzlicher Verbindungen bzw. deren Reaktionsprodukten mit Proteinen u.a. über massenspektrometrische Methoden. Da die Wechselwirkung mit Proteinen auch zu Veränderungen der Proteinstruktur führt, können deren funktionelle Eigenschaften auch verändert sein. Dies soll anhand der Messung von isolierten Proteinen die an der Wechselwirkung beteiligt sind, nachgewiesen werden. Anschließend sollen über Docking-Untersuchungen die entsprechenden Bindungsstellen näher charakterisiert werden. Durch die vorliegenden Ergebnisse wurden mögliche Reaktionen von phenolischen Verbindungen mit Proteinen, näher charakterisiert. Es wurde festgestellt, dass die Apfelsorte Braeburn über die höchste PPO- Enzymaktivität beim gleichzeitigen niedrigen CQA Gehalt im Vergleich zu den anderen untersuchten Sorten verfügt. Die PPO/Tyrosinase modulierte Reaktionen zwischen CQA und Lysine wurden in Abhängigkeit der vorherrschenden Bedingungen optimiert und die Reaktionsprodukte analysiert. In dem zweiten Teil wurden solche Reaktionsmöglichkeiten in den Grünen Kaffeebohnen lokalisierte und modelliert. Dazu wurden die sortenabhängige CQA-Zusammensetzung ermittelt und die möglichen Reaktionen mit den Hauptspeicherproteinen des Kaffees dargestellt. Im letzten Teil wurden dann diese Reaktionen mit Molkenproteinen simuliert und Einflüsse auf die Struktur und die funktionellen Eigenschaften erfasst. Die Ergebnisse belegen eine umfangreiche und sehr heterogene Adduktbildung mit den Aminoseitenketten des Lysins und Cysteins. Ein Katalog der unterschiedlichen Reaktionsprodukte wurde erstellt und am Protein modelliert. Die entsprechende Veränderung an die Proteinstruktur wurde experimentell belegt und der Einfluss wurde in den technofunktionelle Eigenschaften (wie die Löslichkeit, Emulgierbarkeit usw.) wiederspiegelt. Ein Anstieg des antioxidativen Potentials der Proteine wurde erreicht und diese so modifizierten Proteine wurden weiter zur Stabilisierung und Produktentwicklung getestet. Die ersten Ergebnisse eröffnen Nutzungsmöglichkeiten der modifizierten Proteine zur Verkapselung von bioaktiven Sekundären Pflanzenstoffen.

Tea and coffee are rich in polyphenols with a variety of biological activities. Polyphenols found in tea are predominantly flavonoids, of which up to 15% are present as free or esterified gallic acid. Coffee polyphenols are almost wholly comprised of chlorogenic acids. Many of the demonstrated activities of polyphenols are consistent with favourable effects on the risk of chronic diseases. In investigating the relationships between intake and exposure to such compounds and chronic disease-related endpoints, it is important to be able to identify biomarkers that are specific to the compounds of interest. 4-O-methyl gallic acid (4OMGA) and isoferulic acid have been identified as potential biomarkers of intake and exposure to polyphenols derived from tea and coffee, respectively. 4OMGA is derived from gallic acid in tea, and isoferulic acid from chlorogenic acid in coffee. The major objectives of the research which is the subject of this thesis were (1) to establish a dose-response relationship of 24h urinary excretions of 4OMGA and isoferulic acid following ingestions of black tea and coffee of different strengths, and (2) to explore relationships of tea and coffee intake with 24h urinary excretion of 4OMGA and isoferulic acid in human populations. It was found that there was rapid excretion of both 4OMGA and isoferulic acid in the first 6h after tea and coffee ingestion, respectively. Approximately 60 80% of the ingested dose was excreted during the first 6h after ingestion. Urinary excretion of 4OMGA and isoferulic acid was directly related to the dose of tea and coffee, respectively. That is, higher intake resulted in increased urinary excretion of the metabolites. The relationships of 24h urinary excretion of 4OMGA and isoferulic acid with long-term usual (111 participants) and contemporary recorded current (344 participants) tea and coffee intake were assessed. 4OMGA was strongly related to usual (r = 0.50, P < 0.001) and current (r = 0.57, P < 0.001) tea intake. Isoferulic acid was less strongly, but significantly associated with usual (r = 0.26, P = 0.008) and current (r = 0.18, P < 0.001) coffee intake. Overall, the results are consistent with the proposal that 4OMGA is a good biomarker for black tea derived polyphenol intake and exposure, but isoferulic acid may have only limited use as a biomarker for coffee-derived polyphenol exposure.

This master´s thesis focuses at bioactive compounds in edible berries during its culinary processing. Elderberries (Sambucus nigra L.) were selected as a representative of edible berries variety. Elderberry is known for presence of significant bioactive compound content and usage in traditional medicine. Elderberries were processed acording to common culinary recipes. Amount od selected bioactive polyphenolic compounds (chlorogenic acid, quercetin, rutin, anthocyanes) was detected accordingly. Additionally content of ascorbic acid was determined. On the basis of the experimental determination, it was found that anthocyanes and rutin are the most common compounds found in elderberries. All analytes in juices and sirups gradually decreased during the treatments and long-term storage. On the contrary, the content of quercetin increased with gradual modifications and storage. The highest decrease was observed for vitamin C content. The lowest values in the products from elderberries were reached by quercetin and ascorbic acid.

Observa-se, atualmente, um aumento no interesse por alimentos funcionais e isto tem levado pesquisadores a investigar o potencial antioxidante de diversas frutas e de diversos compostos fenólicos presentes nos alimentos. As pesquisas a cerca da atividade antioxidante de pêssegos e nectarinas são escassas quando comparada às frutas vermelhas, apesar de aqueles frutos conterem importantes compostos fenólicos, como o ácido clorogênico, que apresenta efeitos potencialmente benéficos à saúde derivados de sua ação antioxidante. Entretanto, dados a respeito do seu conteúdo em frutas, vegetais, alimentos processados e sua distribuição em diferentes tecidos de plantas são escassos. O potencial antioxidante reativo total (TRAP) é um dos métodos mais utilizados para estimar a capacidade antioxidante de amostras in vitro. Entretanto, este método apresenta limitações quando a amostra não apresenta a fase lag (ou tempo de indução) necessária para obter o valor do TRAP pelo método original. Esse comportamento é obtido em algumas amostras e em substâncias isoladas, mas não em muitos extratos, os quais possuem diversas substâncias antioxidantes. Nesse contexto, o objetivo desse trabalho foi otimizar e validar o método TRAP original e propor outro método de avaliação utilizando a Área Sob a Curva (AUC) e empregar essa ferramenta para avaliar o potencial antioxidante de extratos de pêssego (cascas e polpas) de três cultivares e do ácido clorogênico na mesma concentração encontrada no extrato. Para desenvolver esse trabalho, o primeiro passo foi otimizar e validar o método TRAP alternativo utilizando 2,2'-azo-bis (2- amidinopropano) (AAPH) como fonte de radical livre e um cintilador líquido para monitorar a quimiluminescência amplificada pelo luminol após a adição da amostra antioxidante. O método cromatográfico para quantificar o ácido clorogênico foi realizado usando uma coluna C-18 e sistema gradiente de eluição com acetonitrila-água-ácido trifluoracético. A detecção foi realizada por ultravioleta a 327 nm e a identificação do pico foi baseada no tempo de retenção, por coinjeções com a substância de referência e por LC-MS-MS. A principal condição estabelecida para o método TRAP foi a necessidade de estabilização do sistema (7000 segundos) antes da adição do antioxidante a ser testado. Todos os parâmetros da validação mostraram resultados satisfatórios: especificidade, linearidade (r > 0,99), precisão (intra e inter-dia – desvio padrão relativo menos que 5%), robustez e os limites de detecção (LOD) e quantificação (LOQ) foram baixos e similares para ambos os métodos de avaliação. O método cromatográfico para quantificar o ácido clorogênico apresentou boa linearidade (r > 0,99), repetibilidade (RSD < 2%) e recuperação (96,3%, RSD = 1,96%) e foi robusto para pequenas variações nos parâmetros testados (RSD < 2%). Os resultados obtidos usando a AUC mostraram que os extratos de cascas e polpas das cultivares Maciel, Leonense e Eldorado apresentaram atividade de seqüestro de radicais livres em todas as concentrações testadas, com uma ação concentraçãodependente. A inibição imediata da quimiluminescência e a duração desta inibição foram significativamente maiores no extrato em relação ao composto majoritário (ácido clorogênico) isolado e isto pode ser devido a um efeito sinérgico ou aditivo de outros antioxidantes presentes no extrato. O IC50 para o extrato de pêssego e ácido clorogênico foi 1,19 μg/mL e 8,43 μg/mL, respectivamente, quando o TRAP foi avaliado e um IC50 de 0,41 μg/mL e 1,89 μg/mL foi obtido quando o TAR (Reatividade Antioxidante Total) foi avaliado com o extrato de pêssego e com o ácido clorogênico, respectivamente. O ácido clorogênico apresentou uma importante contribuição ao potencial e à reatividade antioxidantes, mas os extratos do fruto contribuem com uma ação antioxidante mais duradoura. A principal vantagem da utilização da AUC para avaliar a atividade antioxidante é a maior precisão desse método e seu uso em amostras que não apresentam fase lag, como os extratos de pêssego, os quais apresentaram importante atividade antioxidante e, portanto, podem proporcionar vantagens à saúde de consumidores que ingerem essa fruta. / Increasing recent interest in nutraceuticals and functional foods has led researchers to investigate the antioxidant potential of several fruits and of several phenolic compounds. The researches about antioxidant activity of peaches and nectarines are scarce when compared to red fruits despite of they contain important phenolic compounds as chlorogenic acid, which present potential beneficial effect in humans derived from its antioxidant activity. However, data on its content in fruits, vegetables, foods processing, and its distribution in different tissues of plants is scarce. The total reactivity antioxidant potencial (TRAP) method is one of the methods most employed to estimate the antioxidant capacity of samples in vitro. However, this method can presents limitations when the sample does not present a lag phase (or induction time) which is necessary to get TRAP value by original method. This behavior is got in some samples and to isolated substances, but not in many extracts which have several antioxidants. In this context, the aim of this work was to optimize and validate the original TRAP method, and to propose another evaluation method utilizing the area under curve (AUC) and to use this tool to evaluate antioxidant potential and reactivity from peach extracts (peels and fleshes) from three cultivars and from chlorogenic acid in the same concentration founded in extract. To developed this work, the first step was to optimized and to validated the alternative method to get TRAP value using 2,2'-azo-bis (2-amidinopropane) (AAPH) as the free radical source and a liquid scintillator counter to monitored the luminol-enhanced chemiluminescence after addition of the antioxidant sample. The chromatographic method to quantifiy chlorogenic acid was performed using a C-18 column with acetonitrile-watertrifluoracetic acid gradient elution. The detection was carried out by UV at 327 nm and the peak identification was based on the retention times, by cochromatography with reference substances and by LC-MS-MS. The main condition established to TRAP method was the need for the stabilization of the system, at 7000 s, before the addition of the antioxidant to be tested. All validations parameters showed satisfactory results: specificity, linearity (r >0.99), precision (intra- and interassay - relative standard deviation were both less than 5 %), robustness, and the limits of detection (LOD) and quantification (LOQ) were low and similar to both evaluation methods. The chromatographic method to quantify chlorogenic acid presented good linearity (> 0.99), good repeatability (RSD < 2 %) and accuracy (96.3%, RSD = 1.96% ) and was robust to small variations in parameters tested (RSD < 2 %). The results obtained using AUC showed that the extracts of peels and fleshes of Maciel, Leonense and Eldorado peach cultivars present free radical scavenging activity in all concentrations tested, with a concentration-dependent action. The immediate inhibition of chemiluminescence and the duration of this inhibition were significantly higher in the extracts than in the major compound (chlorogenic acid) alone and it can be due to a synergistic or additive effect of other antioxidants present in the extracts. The IC50 for peach extract and chlorogenic acid were 1.19 μg/mL and 8.43 μg/mL, respectively, when TRAP was evaluated and an IC50 of 0.41 μg/mL and 1.89 μg/mL was found when TAR was evaluated in peach extract and chlorogenic acid, respectively. Chlorogenic acid presented a good contribution to antioxidant reactivity and potential, but the fruit extracts provide better antioxidant action. The main advantage of utilization of AUC to evaluate antioxidant activity is the higher precision of this method and its use in samples that do not present lag phase, as peach extracts which presented important antioxidant activity and therefore, it may provide health-promoting advantages to consumers by intake of this fruit.

O pêssego é o nome popular do fruto produzido por Prunus persica (L.) Batsch, e é amplamente cultivado na região sul do Brasil. Seu cultivo é economicamente muito importante para o local, e o desenvolvimento de técnicas que garantam o controle de qualidade desta matéria-prima e seus produtos industriais são interessantes. No presente trabalho foi feita uma revisão de literatura acerca de análises realizadas por CLAE-UV com o pêssego, realizada uma otimização das técnicas de extração do ácido clorogênico e do sistema cromatográfico, validado este método e utilizado para quantificar 13 diferentes cultivares de pêssegos fornecidos pela EMBRAPA Clima Temperado, cultivados em Pelotas, RS. O resultado é um método rápido, econômico e prático que foi otimizado e validado, excelente para análises rotineiras de controle de qualidade deste material. As quantidades de ácido clorogênico foram comparadas entre si, e identificou-se que as cultivares Morro Redondo e Sunblaze apresentaram os maiores teores desta substância, sendo promissoras para serem empregadas pela indústria como alimento funcional. / Peach is the common name of the fruit produced by Prunus persica (L.) Batsch, widely cultivated in the southern of Brazil. Its cultivation is economically important to the local market, and the development of techniques that assure the quality control of the raw material as well as the industrial products made of this fruit are very useful. In this work, a literature review of the HPLC-UV analysis of peach was done, a technique of extraction and chromatographic system were optimized and validated, and 13 cultivar varieties supplied by EMBRAPA Clima Temperado, grown in Pelotas, RS were analyzed. The result is an optimized and validated method of HPLC-UV to quantify chlorogenic acid in peaches, excellent for routine analysis. The average amount of this phenolic compound in the selected cultivars was quantified and compared among them. It was identified that Morro Rendondo and Sunblaze varieties presents the highest amounts of this substance, and both are promising cultivars to be explored by the industry as functional food.

The cardiovascular diseases consist of a public health problem of determined by a complex network of risky factors, with distinction to those related to the life style. Coffee consumption has been pointed out as one of these factors, based on the proposal of which the diterpenes there present would raise the cholesterol and serum triglycerides, one of the most important factors of cardiovascular risk. In this context, objectifying to contribute in the discussion of this problem in focus, this dissertation presents two articles: a revision of literature concerning substances present in the coffee drink and its relation to the systemic arterial and dyslipidemias hypertension, and an original article that presents a transversal study where the association between the consumption of coffee and serum lipid profile in hypertensions and diabetics type 2 were verified. Studies on the identification of substances present in coffee and the relation of many of them to the health have been growing in the last few decades, which is understood because of the fact that coffee is one of most popular drinks, world-widely consumed, as well as for its economic importance in Brazil and in the European countries. The caffeine, mainly the cafestol and kahweol diterpenes and the chlorogenic acids are the composites most discussed in this dissertation. Emphasis is given to the preparation methods of the coffee drink, consumption and dilution, and the possible relation of these factors with the serum lipid profile and dyslipidemias. / Fundação de Amparo a Pesquisa do Estado de Alagoas / As doenças cardiovasculares constituem um problema de saúde pública determinado por uma complexa rede de fatores de risco, com destaque para aqueles relacionados ao estilo de vida. O consumo de café tem sido apontado como um destes fatores, baseado na proposição de que os diterpenos ali presentes elevariam o colesterol e triglicerídeos séricos, um dos mais importantes fatores de risco cardiovascular. Neste contexto, objetivando contribuir na discussão deste problema em foco, esta dissertação apresenta dois artigos: uma revisão da literatura acerca das substâncias presentes na bebida café e a sua relação com a hipertensão arterial sistêmica e dislipidemias e um artigo original que apresenta um estudo transversal onde foi verificada a associação entre consumo de café e perfil lipídico sérico em hipertensos e diabéticos tipo 2 . Estudos de identificação de substâncias presentes no café e da relação de muitas delas com a saúde vêm crescendo nas últimas décadas, o que é explicado pelo fato do café ser uma das bebidas mais populares, mundialmente consumida, bem como pela sua importância econômica no Brasil e no mundo ocidental. A cafeína, principalmente os diterpenos cafestol e kahweol e os ácidos clorogênicos são os compostos mais discutidos nesta dissertação. Ênfase é dada aos métodos de preparo da bebida café, consumo e diluição e, a possível relação destes fatores com o perfil lipídico sérico e dislipidemias.

O silverskin é um resíduo, rico em compostos antioxidantes, como o ácido 5-cafeoilquínico (5-CQA) e a cafeína (CAF), produzido durante a torrefação dos grãos de café. O objetivo deste estudo foi desenvolver uma metodologia de extração, aliando a extração assistida por ultrassom e o planejamento de experimentos, para a recuperação simultânea do 5-CQA e da CAF da silverskin. Os extratos gerados foram caracterizados e tiveram a capacidade antioxidante e a toxicidade investigadas. A metodologia de superfície de resposta e a função Desejabilidade foram empregadas na otimização da extração. A LC-DAD/MSn foi utilizada para a caracterização dos extratos, os métodos de sequestro do radical DPPH e de redução do ferro (FRAP), para avaliar a capacidade antioxidante; enquanto o ensaio da Artemia salina foi usando para avaliar a toxicidade. As condições ótimas de extração foram: 45 % de etanol em água como solvente de extração, razão amostra-solvente 1/20, extração por 7 min a 59 °C, com rendimentos de 5-ACQ e CAF de respectivamente 2,00 e 6,26 mg g-1. No total, 9 derivados clorogênicos foram tentativamente identificados. Os teores de teofilina, ácido cafeico e derivados clorogênicos foram respectivamente: 0,53, 0,06 e 4,40 mg g-1. O extrato demonstrou considerável capacidade antioxidante, com concentração eficiente de 46,87 mg L-1 e capacidade redutora de ferro de 152,71 μM de FeSO4 g-1, além de alta toxicidade. O método desenvolvido obteve rendimento similar ao da extração sólido-líquido para o 5-ACQ. / Silverskin is a waste generated during the coffee beans roasting. This residue has been showing antioxidant compounds, mainly 5-caffeoylquinic acid (5-CQA) and caffeine (CAF). The aim of this study was used the design of experiments and ultrasound-assisted extraction to develop an extraction method for the efficient recovery of 5-CQA and CAF from silverskin, simultaneously. In addition, the optimized extract was characterized, and its antioxidant capacity and acute toxicity were investigated. The response surface methodology and Desirability function were employed to optimize the extraction. The chemical characterization was carry out by LC-DAD/ESI-MSn. Antioxidant capacity was evaluated by DPPH (2, 2-diphenyl-1 picryl hydrazyl) and FRAP (ferric reducing antioxidant power) methods and the acute toxicity by Artemia salina assay. The optimal extraction conditions were 45.0 % ethanol in water, the solid-to-liquid ratio was 1:20, and extraction for 7.0 min at 59 °C. Under optimal conditions, the yield of 5-CQA and CAF were 2.00 and 6.26 mg g-1, respectively. Theophylline, caffeic acid, and total chlorogenic acid concentrations were 0.53, 0.06 e 4.40 mg g-1, respectively and 9 chlorogenic acids were identified in the extract. The extract showed considerable antioxidant capacity, with efficient concentration value of 46,87 mg L-1 in the DPPH assay and 152,71 μM FeSO4 g-1 (FRAP) and high toxicity. The developed extraction methodology showed similar performance as solid-liquid extraction to 5-CQA yield.